RNA editing in trypanosomes is a post-transcriptional process that results in the formation of mitochondrial mRNAs differing from their genes by the insertion or deletion of uridylyl nucleosides. Small guide RNAs (gRNA) base pair with their cognate pre-edited mRNAs to direct the precise sites for U-insertion and U-deletion. RNA editing is physiologically significant since editing often occurs within the coding portion of mRNAs and results in the creation of long open reading frames encoding essential mitochondrial proteins. RNA editing may also play a role in the developmental regulation of mitochondrial biogenesis since both the extent and pattern of editing correlates with mitochondrial changes during the trypanosome development. Furthermore, recent studies indicate that RNA editing is essential to the bloodstream forms of African trypanosomes suggesting that RNA editing might be a novel target for chemotherapy. Remarkable progress has been made in the identification of many of the enzymes and accessory proteins required for RNA editing. We have proposed that these proteins assemble with pre-edited mRNAs and gRNAs to form a high molecular weight ribonucleoprotein particle (RNP). The overall goal of this proposal is to dissect the pathway of assembly and disassembly of the "editosome" in order to gain a comprehensive understanding of the mechanism and regulation of RNA editing. To accomplish this goal the following specific aims are proposed. In Specific Aim 1, the protein composition of the editosome will be determined using biochemical and genetic approaches and the role of each protein in RNA editing established by RNA interference or gene-disruption. In Specific Aim II, we will determine how RNA editing proceeds along the pre-mRNA often using many different gRNAs-and resulting in the insertion and deletion of hundreds of uridylyl nucleotides. In these studies we will use a newly developed in vivo RNA import system to introduce modified editing substrates and gRNAs into the mitochondrion. In Specific Aim III, we propose a number of experiments to determine whether mitochondrial mRNA and gRNA transcription are coupled to RNA editing. Using pulse chase and immunoprecipitation analysis the physical interactions of the mitochondrial RNA polymerase and editing complex proteins will be established. Together these studies will provide a better understanding of the role of RNPs in RNA editing and the relationship of RNA editing to transcription and other RNA processing events.